Low-dose Dasatinib Ameliorates Hypertrophic Cardiomyopathy in Noonan Syndrome with Multiple Lentigines.

PURPOSE: Noonan syndrome with multiple lentigines (NSML) is an autosomal dominant disorder presenting with hypertrophic cardiomyopathy (HCM). Up to 85% of NSML cases are caused by mutations in the PTPN11 gene that encodes for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2). We previously showed that low-dose dasatinib protects from the development of cardiac fibrosis in a mouse model of NSML harboring a Ptpn11Y279C mutation. This study is performed to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of a low-dose of dasatinib in NSML mice and to determine its effectiveness in ameliorating the development of HCM. METHODS: Dasatinib was administered intraperitoneally into NSML mice with doses ranging from 0.05 to 0.5 mg/kg. PK parameters of dasatinib in NSML mice were determined. PD parameters were obtained for biochemical analyses from heart tissue. Dasatinib-treated NSML mice (0.1 mg/kg) were subjected to echocardiography and assessment of markers of HCM by qRT-PCR. Transcriptome analysis was performed from the heart tissue of low-dose dasatinib-treated mice. RESULTS: Low-dose dasatinib exhibited PK properties that were linear across doses in NSML mice. Dasatinib treatment of between 0.05 and 0.5 mg/kg in NSML mice yielded an exposure-dependent inhibition of c-Src and PZR tyrosyl phosphorylation and inhibited AKT phosphorylation. We found that doses as low as 0.1 mg/kg of dasatinib prevented HCM in NSML mice. Transcriptome analysis identified differentially expressed HCM-associated genes in the heart of NSML mice that were reverted to wild type levels by low-dose dasatinib administration. CONCLUSION: These data demonstrate that low-dose dasatinib exhibits desirable therapeutic PK properties that is sufficient for effective target engagement to ameliorate HCM progression in NSML mice. These data demonstrate that low-dose dasatinib treatment may be an effective therapy against HCM in NSML patients.

Generation of a hypoxia-sensing mouse model.

Oxygen (O2 ) homeostasis is essential to the metazoan life. O2 -sensing or hypoxia-regulated molecular pathways are intimately involved in a wide range of critical cellular functions and cell survival from embryogenesis to adulthood. In this report, we have designed an innovative hypoxia sensor (O2 CreER) based on the O2 -dependent degradation domain of the hypoxia-inducible factor-1α and Cre recombinase. We have further generated a hypoxia-sensing mouse model, R26-O2 CreER, by targeted insertion of the O2 CreER-coding cassette in the ROSA26 locus. Using the ROSAmTmG mouse strain as a reporter, we have found that this novel hypoxia-sensing mouse model can specifically identify hypoxic cells under the pathological condition of hind-limb ischemia in adult mice. This model can also label embryonic cells including vibrissal follicle cells in E13.5-E15.5 embryos. This novel mouse model offers a valuable genetic tool for the study of hypoxia and O2 sensing in mammalian systems under both physiological and pathological conditions.

Polycystin 2-dependent cardio-protective mechanisms revealed by cardiac stress.

Although autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple kidney cysts, the most frequent cause of death in ADPKD patients is cardiovascular disease. ADPKD is linked to mutations in PKD1 or pkd2, the genes that encode for the proteins polycystin 1 and polycystin 2 (PC1 and PC2, respectively). The cardiovascular complications have been assumed to be a consequence of renal hypertension and activation of renin/angiotensin/aldosterone (RAAS) pathway. However, the expression of PC1 and PC2 in cardiac tissue suggests additional direct effects of these proteins on cardiac function. We previously reported that zebrafish lacking PC2 develop heart failure, and that heterozygous Pkd2+/- mice are hypersensitive to acute ß-adrenergic receptor (ßAR) stimulation. Here, we investigate the effect of cardiac stress (prolonged continuous ßAR stimulus) on Pkd2+/- mice. After ßAR stimulation for 7 days, wild-type (WT) mice had increased left ventricular mass and natriuretic peptide (ANP and BNP) mRNA levels. The WT mice also had upregulated levels of PC2 and chromogranin B (CGB, an upstream regulator of BNP). Conversely, Pkd2+/- mice had increased left ventricular mass, but natriuretic peptide and CGB expression levels remained constant. Reversal of the increased cardiac mass was observed in WT mice 3 days after cessation of the ßAR stimulation, but not in Pkd2+/- mice. We suggest that cardiac stress leads to upregulation of the PC2-CGB-BNP signaling axis, and this pathway regulates the production of cardio-protective natriuretic peptides. The lack of a PC2-dependent cardio-protective function may contribute to the severity of cardiac dysfunction in Pkd2+/- mice and in ADPKD patients.

Endothelial HIF-1α Enables Hypothalamic Glucose Uptake to Drive POMC Neurons.

Glucose is the primary driver of hypothalamic proopiomelanocortin (POMC) neurons. We show that endothelial hypoxia-inducible factor 1α (HIF-1α) controls glucose uptake in the hypothalamus and that it is upregulated in conditions of undernourishment, during which POMC neuronal activity is decreased. Endothelium-specific knockdown of HIF-1α impairs the ability of POMC neurons to adapt to the changing metabolic environment in vivo, resulting in overeating after food deprivation in mice. The impaired functioning of POMC neurons was reversed ex vivo or by parenchymal glucose administration. These observations indicate an active role for endothelial cells in the central control of metabolism and suggest that central vascular impairments may cause metabolic disorders.

Low-dose dasatinib rescues cardiac function in Noonan syndrome.

Noonan syndrome (NS) is a common autosomal dominant disorder that presents with short stature, craniofacial dysmorphism, and cardiac abnormalities. Activating mutations in the PTPN11 gene encoding for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) causes approximately 50% of NS cases. In contrast, NS with multiple lentigines (NSML) is caused by mutations that inactivate SHP2, but it exhibits some overlapping abnormalities with NS. Protein zero-related (PZR) is a SHP2-binding protein that is hyper-tyrosyl phosphorylated in the hearts of mice from NS and NSML, suggesting that PZR and the tyrosine kinase that catalyzes its phosphorylation represent common targets for these diseases. We show that the tyrosine kinase inhibitor, dasatinib, at doses orders of magnitude lower than that used for its anticancer activities inhibited PZR tyrosyl phosphorylation in the hearts of NS mice. Low-dose dasatinib treatment of NS mice markedly improved cardiomyocyte contractility and functionality. Remarkably, a low dose of dasatinib reversed the expression levels of molecular markers of cardiomyopathy and reduced cardiac fibrosis in NS and NSML mice. These results suggest that PZR/SHP2 signaling is a common target of both NS and NSML and that low-dose dasatinib may represent a unifying therapy for the treatment of PTPN11-related cardiomyopathies.

Endothelial Nogo-B regulates sphingolipid biosynthesis to promote pathological cardiac hypertrophy during chronic pressure overload.

We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α-activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B-deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition.

Smooth Muscle Hypoxia-Inducible Factor 1α Links Intravascular Pressure and Atherosclerosis--Brief Report.

OBJECTIVE: We hypothesized that the hypoxia-inducible factor (HIF) 1α in vascular smooth muscle contributes to the development of atherosclerosis, and links intravascular pressure to this process. APPROACH AND RESULTS: Transverse aortic constriction was used to create high-pressure vascular segments in control, apolipoprotein E (ApoE)(-/-), smooth muscle-HIF1α(-/-), and ApoE(-/-)×smooth muscle-HIF1α(-/-) double-knockout mice. Transverse aortic constriction selectively induced atherosclerosis in high-pressure vascular segments in young ApoE(-/-) mice on normal chow, including coronary plaques within 1 month. Concomitant deletion of HIF1α from smooth muscle significantly reduced vascular inflammation, and attenuated atherosclerosis. CONCLUSIONS: HIF1α in vascular smooth muscle plays an important role in the pathogenesis of atherosclerosis, and may provide a mechanistic link between blood pressure, vascular inflammation, and lipid deposition.

Interferon-γ-mediated allograft rejection exacerbates cardiovascular disease of hyperlipidemic murine transplant recipients.

RATIONALE: Transplantation, the most effective therapy for end-stage organ failure, is markedly limited by early-onset cardiovascular disease (CVD) and premature death of the host. The mechanistic basis of this increased CVD is not fully explained by known risk factors. OBJECTIVE: To investigate the role of alloimmune responses in promoting CVD of organ transplant recipients. METHODS AND RESULTS: We established an animal model of graft-exacerbated host CVD by combining murine models of atherosclerosis (apolipoprotein E-deficient recipients on standard diet) and of intra-abdominal graft rejection (heterotopic cardiac transplantation without immunosuppression). CVD was absent in normolipidemic hosts receiving allogeneic grafts and varied in severity among hyperlipidemic grafted hosts according to recipient-donor genetic disparities, most strikingly across an isolated major histocompatibility complex class II antigen barrier. Host disease manifested as increased atherosclerosis of the aorta that also involved the native coronary arteries and new findings of decreased cardiac contractility, ventricular dilatation, and diminished aortic compliance. Exacerbated CVD was accompanied by greater levels of circulating cytokines, especially interferon-γ and other Th1-type cytokines, and showed both systemic and intralesional activation of leukocytes, particularly T-helper cells. Serological neutralization of interferon-γ after allotransplantation prevented graft-related atherosclerosis, cardiomyopathy, and aortic stiffening in the host. CONCLUSIONS: Our study reveals that sustained activation of the immune system because of chronic allorecognition exacerbates the atherogenic diathesis of hyperlipidemia and results in de novo cardiovascular dysfunction in organ transplant recipients.

Nogo-B regulates endothelial sphingolipid homeostasis to control vascular function and blood pressure.

Endothelial dysfunction is a critical factor in many cardiovascular diseases, including hypertension. Although lipid signaling has been implicated in endothelial dysfunction and cardiovascular disease, specific molecular mechanisms are poorly understood. Here we report that Nogo-B, a membrane protein of the endoplasmic reticulum, regulates endothelial sphingolipid biosynthesis with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, thereby controlling production of endothelial sphingosine 1-phosphate and autocrine, G protein-coupled receptor-dependent signaling by this metabolite. Mice lacking Nogo-B either systemically or specifically in endothelial cells are hypotensive, resistant to angiotensin II-induced hypertension and have preserved endothelial function and nitric oxide release. In mice that lack Nogo-B, pharmacological inhibition of serine palmitoyltransferase with myriocin reinstates endothelial dysfunction and angiotensin II-induced hypertension. Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and shows that autocrine sphingolipid signaling within the endothelium is critical for vascular function and blood pressure homeostasis.

Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function.

BACKGROUND: Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. METHODS AND RESULTS: Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation. CONCLUSIONS: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

Activation of hypoxia-inducible factor-2 in adipocytes results in pathological cardiac hypertrophy.

BACKGROUND: Obesity can cause structural and functional abnormalities of the heart via complex but largely undefined mechanisms. Emerging evidence has shown that obesity results in reduced oxygen concentrations, or hypoxia, in adipose tissue. We hypothesized that the adipocyte hypoxia-signaling pathway plays an essential role in the development of obesity-associated cardiomyopathy. METHODS AND RESULTS: Using a mouse model in which the hypoxia-inducible factor (HIF) pathway is activated by deletion of the von Hippel-Lindau gene specifically in adipocytes, we found that mice with adipocyte-von Hippel-Lindau deletion developed lethal cardiac hypertrophy. HIF activation in adipocytes results in overexpression of key cardiomyopathy-associated genes in adipose tissue, increased serum levels of several proinflammatory cytokines including interleukin-1ß and monocyte chemotactic protein-1, and activation of nuclear factor-κB and nuclear factor of activated T cells in the heart. Interestingly, genetic deletion of Hif2a, but not Hif1a, was able to rescue cardiac hypertrophy and abrogate adipose inflammation. CONCLUSION: We have discovered a previously uncharacterized mechanism underlying a critical and direct role of the adipocyte HIF-2 transcription factor in the development of adipose inflammation and pathological cardiac hypertrophy.

Hypoxia-inducible factor-1α in vascular smooth muscle regulates blood pressure homeostasis through a peroxisome proliferator-activated receptor-γ-angiotensin II receptor type 1 axis.

Hypertension is a major worldwide health issue for which only a small proportion of cases have a known mechanistic pathogenesis. Of the defined causes, none have been directly linked to heightened vasoconstrictor responsiveness, despite the fact that vasomotor tone in resistance vessels is a fundamental determinant of blood pressure. Here, we reported a previously undescribed role for smooth muscle hypoxia-inducible factor-1α (HIF-1α) in controlling blood pressure homeostasis. The lack of HIF-1α in smooth muscle caused hypertension in vivo and hyperresponsiveness of resistance vessels to angiotensin II stimulation ex vivo. These data correlated with an increased expression of angiotensin II receptor type I in the vasculature. Specifically, we show that HIF-1α, through peroxisome proliferator-activated receptor-γ, reciprocally defined angiotensin II receptor type I levels in the vessel wall. Indeed, pharmacological blockade of angiotensin II receptor type I by telmisartan abolished the hypertensive phenotype in smooth muscle cell-HIF-1α-KO mice. These data revealed a determinant role of a smooth muscle HIF-1α/peroxisome proliferator-activated receptor-γ/angiotensin II receptor type I axis in controlling vasomotor responsiveness and highlighted an important pathway, the alterations of which may be critical in a variety of hypertensive-based clinical settings.

Rapamycin inhibits smooth muscle cell proliferation and obstructive arteriopathy attributable to elastin deficiency.

OBJECTIVE: Patients with elastin deficiency attributable to gene mutation (supravalvular aortic stenosis) or chromosomal microdeletion (Williams syndrome) are characterized by obstructive arteriopathy resulting from excessive smooth muscle cell (SMC) proliferation, mural expansion, and inadequate vessel size. We investigated whether rapamycin, an inhibitor of the cell growth regulator mammalian target of rapamycin (mTOR) and effective against other SMC proliferative disorders, is of therapeutic benefit in experimental models of elastin deficiency. APPROACH AND RESULTS: As previously reported, Eln(-/-) mice demonstrated SMC hyperplasia and severe stenosis of the aorta, whereas Eln(+/-) mice exhibited a smaller diameter aorta with more numerous but thinner elastic lamellae. Increased mTOR signaling was detected in elastin-deficient aortas of newborn pups that was inhibited by maternal administration of rapamycin. mTOR inhibition reduced SMC proliferation and aortic obstruction in Eln(-/-) pups and prevented medial hyperlamellation in Eln(+/-) weanlings without compromising aortic size. However, rapamycin did not prolong the survival of Eln(-/-) pups, and it retarded the somatic growth of juvenile Eln(+/-) and Eln(+/+) mice. In cell cultures, rapamycin inhibited prolonged mTOR activation and enhanced proliferation of SMC derived from patients with supravalvular aortic stenosis and with Williams syndrome. CONCLUSIONS: mTOR inhibition may represent a pharmacological strategy to treat diffuse arteriopathy resulting from elastin deficiency.

IL-13 receptor α2-arginase 2 pathway mediates IL-13-induced pulmonary hypertension.

Although previous literature suggests that interleukin (IL)-13, a T-helper type 2 cell effector cytokine, might be involved in the pathogenesis of pulmonary hypertension (PH), direct proof is lacking. Furthermore, a potential mechanism underlying IL-13-induced PH has never been explored. This study's goal was to investigate the role and mechanism of IL-13 in the pathogenesis of PH. Lung-specific IL-13-overexpressing transgenic (Tg) mice were examined for hemodynamic changes and pulmonary vascular remodeling. IL-13 Tg mice spontaneously developed PH phenotype by the age of 2 mo with increased expression and activity of arginase 2 (Arg2). The role of Arg2 in the development of IL-13-stimulated PH was further investigated using Arg2 and IL-13 receptor α2 (Rα2) null mutant mice and the small-interfering RNA (siRNA)-silencing approach in vivo and in vitro, respectively. IL-13-stimulated medial thickening of pulmonary arteries and right ventricle systolic pressure were significantly decreased in the IL-13 Tg mice with Arg2 null mutation. On the other hand, the production of nitric oxide was further increased in the lungs of these mice. In our in vitro evaluations, the recombinant IL-13 treatment significantly enhanced the proliferation of human pulmonary artery smooth muscle cells in an Arg2-dependent manner. The IL-13-stimulated cellular proliferation and the expression of Arg2 in hpaSMC were markedly decreased with IL-13Rα2 siRNA silencing. Our studies demonstrate that IL-13 contributes to the development of PH via an IL-13Rα2-Arg2-dependent pathway. The intervention of this pathway could be a potential therapeutic target in pulmonary arterial hypertension.

Normal glucose uptake in the brain and heart requires an endothelial cell-specific HIF-1α-dependent function.

Although intimately positioned between metabolic substrates in the bloodstream and the tissue parenchymal cells that require these substrates, a major role of the vascular endothelium in the regulation of tissue metabolism has not been widely appreciated. We hypothesized that via control of transendothelial glucose transport and contributing paracrine mechanisms the endothelium plays a major role in regulating organ and tissue glucose metabolism. We further hypothesized that the hypoxia-inducible factor -1α (HIF-1α) plays an important role in coordinating these endothelial functions. To test these hypotheses, we generated mice with endothelial cell-specific deletion of HIF-1α. Loss of HIF in the endothelium resulted in significantly increased fasting blood glucose levels, a blunted insulin response with delayed glucose clearance from the blood after i.v. loading, and significantly decreased glucose uptake into the brain and heart. Endothelial HIF-1α knockout mice also exhibited a reduced cerebrospinal fluid/blood glucose ratio, a finding consistent with reduced transendothelial glucose transport and a diagnostic criterion for the Glut1 deficiency genetic syndrome. Endothelial cells from these mice demonstrated decreased Glut1 levels and reduced glucose uptake that was reversed by forced expression of Glut1. These data strongly support an important role of the vascular endothelium in determining whole-organ glucose metabolism and indicate that HIF-1α is a critical mediator of this function.

Cellularity and structure of fresh human coronary thrombectomy specimens; presence of cells with markers of progenitor cells.

Acute coronary syndromes and acute myocardial infarctions are often related to plaque rupture and the formation of thrombi at the site of the rupture. We examined fresh coronary thrombectomy specimens from patients with acute coronary syndromes and assessed their structure and cellularity. The thrombectomy specimens consisted of platelets, erythrocytes and inflammatory cells. Several specimens contained multiple cholesterol crystals. Culture of thrombectomy specimens yielded cells growing in various patterns depending on the culture medium used. Culture in serum-free stem cell enrichment medium yielded cells with features of endothelial progenitor cells which survived in culture for a year. Immunohistochemical analysis of the thrombi revealed cells positive for CD34, cells positive for CD15 and cells positive for desmin in situ, whereas cultured cell from thrombi was desmin positive but pancytokeratin negative. Cells cultured in endothelial cell medium were von Willebrand factor positive. The content of coronary thrombectomy specimens is heterogeneous and consists of blood cells but also possibly cells from the vascular wall and cholesterol crystals. The culture of cells contained in the specimens yielded multiplying cells, some of which demonstrated features of haematopoietic progenitor cells and which differentiated into various cell-types.

Engineered zinc-finger proteins can compensate genetic haploinsufficiency by transcriptional activation of the wild-type allele: application to Willams-Beuren syndrome and supravalvular aortic stenosis.

Williams-Beuren syndrome (WBS) and supravalvular aortic stenosis (SVAS) are genetic syndromes marked by the propensity to develop severe vascular stenoses. Vascular lesions in both syndromes are caused by haploinsufficiency of the elastin gene. We used these distinct genetic syndromes as models to evaluate the feasibility of using engineered zinc-finger protein transcription factors (ZFPs) to achieve compensatory expression of haploinsufficient genes by inducing augmented expression from the remaining wild-type allele. For complex genes with multiple splice variants, this approach could have distinct advantages over cDNA-based gene replacement strategies. Targeting the elastin gene, we show that transcriptional activation by engineered ZFPs can induce compensatory expression from the wild-type allele in the setting of classic WBS and SVAS genetic mutations, increase elastin expression in wild-type cells, induce expression of the major elastin splice variants, and recapitulate their natural stoichiometry. Further, we establish that transcriptional activation of the mutant allele in SVAS does not overcome nonsense-mediated decay, and thus ZFP-mediated transcriptional activation is not likely to induce production of a mutant protein, a crucial consideration. Finally, we show in bioengineered blood vessels that ZFP-mediated induction of elastin expression is capable of stimulating functional elastogenesis. Haploinsufficiency is a common mechanism of genetic disease. These findings have significant implications for WBS and SVAS, and establish that haploinsufficiency can be overcome by targeted transcriptional activation without inducing protein expression from the mutant allele.

A designed zinc-finger transcriptional repressor of phospholamban improves function of the failing heart.

Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.

miR-1 mediated suppression of Sorcin regulates myocardial contractility through modulation of Ca2+ signaling.

MicroRNAs are negative gene regulators and play important roles in cardiac development and disease. As evident by cardiomyopathy following cardiac-specific Dicer knockdown they also are required for maintaining normal cardiac contractile function but the specific role of miR-1 in the process is poorly understood. To characterize the role of miR-1 in particular and to identify its specific targets we created a tamoxifen-inducible, cardiac-specific Dicer knockdown mouse and demonstrated that Dicer downregulation results in a dramatic and rapid decline in cardiac function concurrent with significantly reduced levels of miR-1. The importance of miR-1 was established by miR-1 antagomir treatment of wild-type mice, which replicated the cardiac-specific Dicer knockdown phenotype. Down-regulation of miR-1 was associated with up-regulation of its predicted target Sorcin, an established modulator of calcium signaling and excitation-contraction coupling, subsequently verified as a miR-1 target with luciferase constructs. siRNA-mediated knockdown of Sorcin effectively rescued the cardiac phenotypes after Dicer or miR-1 knockdown affirming Sorcin as a critical mediator of the acute cardiomyopathy observed. The regulatory relationship between miR-1 and Sorcin was further confirmed in cultured mouse cardiomyocytes where modulation of miR-1 was associated with discordant Sorcin levels and dysregulation of calcium signaling. Pathological relevance of our findings included decreased miR-1 and increased Sorcin expression in end-stage cardiomyopathy. These findings demonstrate the importance of miR-1 in cardiac function and in the pathogenesis of heart failure via Sorcin-dependent calcium homeostasis.